Specific identification of Pénicillium marneffei by a polymerase chain reaction/hybridization technique

Pénicillium marneffei has been described recently as a cause of an emerging mycotic infection in HIV-infected patients. A PCR/hybridization assay was developed to rapidly identify this pathogen. The nucleotide sequence of the 631-bp region of 18S ribosomal DNA of P. marneffei was determined using the standard dideoxy chain termination method. An oligonucleotide probe was designed on the basis of the analysed sequences of P. marneffei and 18S rDNA sequences of other fungi in the GenBank database. A 631-bp PCR product was amplified using primers RRF1 and RRH1 from P. marneffei and seven other fungi, Pénicillium spp., Aspergillusfumigatus, A. flavus, Histoplasma capsulatum, Cryptococcus neoformans, Candida albicans and C krusei. A 15 oligonucleotide segment (Pm3) which was specific for P. marneffei was synthesized and used as a probe. Only the PCR products of P. marneffei isolates hybridized with the Pm3 oligonucleotide probe. The sensitivity of the assay was approximately Q-5pg/u\ and 0-1 pg/1 of DNA by PCR and Southern hybridization, respectively. The usefulness of this method as a diagnostic tool will require further studies.